Brilacidin, a novel antifungal agent against Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

virulence determinants, such as the presence of a polysaccharide capsule, the synthesis of melanin, the ability to proliferate at human body temperature and inside of macrophages, tolerance to CO 2 , and the production of host-damaging enzymes like urease and phospholipase (6)(7)(8)(9)(10).These traits contribute to their survival and success as human pathogens (11,12).
The global incidence of cryptococcal meningitis is estimated at more than 400,000 new cases annually, with 181,100 annual deaths (13,14).This number corresponds to approximately 19% of AIDS-related deaths globally (14,15).Despite the significant impact of these infections, treatment options for cryptococcosis remain limited (16,17).The polyenes, azoles, and echinocandins are the three major classes of drugs used for the treatment of invasive fungal infections (18).Polyenes and azoles target ergosterol, depleting the lipid at the fungal plasma membrane or directly blocking its biosynthesis, while the echinocandins act by inhibiting the production of (1,3)-β-D-glucan to disrupt fungal cell wall integrity (16,19).Amphotericin B (AmB), an antifungal of the polyene class, is the first option for the treatment of cryptococcal infections, but it is hampered by substantial toxicity and high cost (20,21).The success of a combination regimen of amphotericin B plus flucytosine [a compound that inhibits pyrimidine biosynthesis (16)] led to updated guidelines for the treatment of cryptococcal disease in HIV patients (20).However, in low-and middle-income countries, the scarcity of these two drugs has resulted in the widespread use of much less effective fluconazole monotherapy, which explains the dramatically high mortality rate (22).
Cryptococcal cells are intrinsically resistant to echinocandins and they employ an arsenal of mechanisms that enable resistance to azoles, such as upregulation of the azole target gene ERG11 (23,24).One is the genomic plasticity that enables cells to form heteroresistant strains under azole stress (25,26).This heteroresistance can be intrinsic and present in all strains regardless of prior drug exposure (27).Genomic plasticity can further enable resistance through the movement of transposable elements, which have been recently linked to drug resistance in C. neoformans (28).Together, these genomic changes allow Cryptococcus spp. a great capacity for physiological adaptation such that they respond quickly and efficiently to stress in the environment and frequently evolve drug resistance (29,30).Other features of C. neoformans, including a complex cell wall, a polysaccharide capsule and the ability to undergo dramatic morphological transitions, contribute to both virulence and drug resistance (31)(32)(33).The plasma membrane also plays an important role in antifungal resistance (34).For example, transporters present in the cell membrane may act as antifungal efflux pumps contributing to azole resistance in C. neoformans and C. gattii (35).Maintenance of lipid asymmetry of the phospholipid membrane is also involved in caspofungin (CAS) resistance (36)(37)(38).
Taking into consideration the increased number of individuals susceptible to cryptococcal infection, the search for new antifungal agents has become more relevant than ever.Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans.BRI is a small molecule host defense peptide mimetic that has previ ously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses (39).Recently, we showed that BRI potentiates the effect of azoles and CAS against several fungal species, decreasing Aspergillus fumigatus fungal burden in a murine chemotherapeutic model of invasive pulmonary aspergillosis and ablating disease development in a murine model of fungal keratitis (40).BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug at concentrations of 2.5 µM, but surprisingly also potentiated the activity of CAS against this species.
Here, we investigated the mechanism of action (MoA) of BRI and BRI + CAS against C. neoformans.We observed that not only C. neoformans but also C. gattii and several clinical isolates from both species are susceptible to BRI, and the combination of BRI + CAS has an additive effect against these strains.We screened a library of C. neofor mans transcription factor mutants and found that deletion of the gene that encodes the sterol regulatory element-binding 1 protein (SRE1) yielded cells showing significant sensitivity to BRI alone.Sre1 is a transcriptional activator required for adaptation to hypoxic growth, which plays an essential role in the ergosterol biosynthesis pathway (41).C. neoformans mutants defective in ergosterol and glycosphingolipid synthesis have reduced minimal inhibitory concentration (MIC) values for BRI and supplementation with free ergosterol reverses the inhibitory effects of BRI, suggesting that this compound affects cell membrane organization.We implicated casein kinases in the BRI MoA, likely via modulating the expression of genes involved in ergosterol biosynthesis and cell wall construction.Analysis of a library of S. cerevisiae mutants grown in the presence of BRI indicated that fungal cell membrane organization, cell wall integrity, and calcium metabolism are affected by this compound.C. neoformans endocytic machinery plays a role in the MoA of BRI + CAS combinations, affecting cell permeability, and the cell wall integrity (CWI) pathway is also important for this MoA, affecting mainly chitin accumu lation in the cell wall.Finally, we showed that BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis.We propose BRI as a new antifungal agent against cryptococcosis.

RESULTS
The MoA of BRI is dependent on the disorganization of the cell membrane in C. neoformans Previously, we have shown that BRI can fungicidally inhibit C. neoformans H99 growth (40).Not only is this strain susceptible to BRI, but C. neoformans KN99a and C. gattii R265, and six clinical isolates, three from each species, are also sensitive to BRI (Table 1).C. neoformans clinical isolates were assayed for mutation rates using a modified Luria-Del bruck fluctuation test (42), for 5-fluorocytosine (5-FC) or BRI (Fig. 1a).Replicate cultures grown without selection were challenged on RPMI medium containing 5-FC or BRI to determine the probability that cells would spontaneously gain mutations that provide resistance (Fig. 1a).On 5-FC, strain H99 and clinical isolate 478 had a spontaneous mutation rate of 1.59 × 10 −8 (95% confidence interval: 6.15 × 10 −9 to 2.56 × 10 −8 ) and 2.37 × 10 −9 (95% confidence interval: 3.61 × 10 −10 to 4.37 × 10 −9 ), respectively, while no BRI-resistant mutants were observed on the RPMI medium containing BRI (Fig. 1a).These results indicate that mutations that confer resistance to BRI were much less likely to occur spontaneously in C. neoformans cells than those that confer resistance to 5-FC.
As an initial step to understand BRI MoA, we screened a C. neoformans H99 transcrip tion factor (TF) null mutants library (43) for susceptibility to BRI.To do this, we grew 322 signature-tagged gene deletion strains, corresponding to 155 putative TF genes, at 30°C on liquid RPMI + BRI (1.25 µM) and then plated them on fresh solid YPD.Two TF mutants, sre1Δ (CNAG_04804) and jjj1Δ (CNAG_05538), were more susceptible to BRI than the wild type (Fig. 1b).One of the sre1Δ mutants showed decreased susceptibility to BRI in solid medium but the same MIC from the wild-type strain in liquid medium (Fig. 1b; Table 1).These two mutants were independently constructed using biolistic bombardment and the differences between them could be due to the introduction of additional mutations in one of them.Sre1p is a homolog of the mammalian sterol regulatory element-bind ing protein (SREBP) that functions in oxygen sensing (44).SRE1 is upregulated in the presence of fluconazole (45), sre1Δ mutants are hypersensitive to azoles, and the protein is required for ergosterol biosynthesis in both hypoxic and normoxic conditions (44).Jjj1p is a DNAJ-like co-chaperone and contains a U1 snRNA-type zinc finger; interestingly, the deletion of the S. cerevisiae JJJ1 homolog causes defects in fluid-phase endocytosis (46).C. neoformans jjj1Δ is more sensitive to AmB and more resistant to fluconazole and ketoconazole than the wild type (47).These results suggested a possible involvement of ergosterol in the BRI MoA.
We hypothesized that the effects observed in ergosterol and sphingolipid mutants could be caused by BRI depolarizing the cell membrane, as in bacterial cells (54).We attempted to determine the effect of BRI on the resting membrane potential using the fluorescent voltage reporter DIBAC 4 (3), but this strategy did not work for C. neoformans.We next tested cell viability using propidium iodide (PI), a fluorescent DNA-binding dye that freely penetrates the cell membranes of dead or dying cells but is excluded from viable cells.When the C. neoformans KN99a cells were incubated without BRI for 4 h, no cells were stained by PI when exposure to increasing BRI concentrations (1.25-25 µM) yielded progressively increased staining (7%-95%) (Fig. 1e).This increased permeability decreased the cell viability from 96% (1.25 µM BRI) to 39% (25 µM BRI) (Fig. 1e; notice the % on the top of the bars represent the cell viability upon exposure to different BRI concentrations).These results suggest that BRI induces C. neoformans permeabilization and cell death.
We speculated if BRI binds directly to ergosterol, the addition of ergosterol to the culture medium would prevent its activity.Ergosterol addition abrogated BRI susceptibil ity completely for C. neoformans H99 and KN99a but to a much lesser extent for C. neoformans sre1Δ and erg3Δ (Fig. 1f).Since these two mutants have reduced ergosterol levels, these results suggest that not all available BRI is binding to free ergosterol.Filipin is an antifungal polyene macrolide that stoichiometrically interacts with sterol to form a stable complex (55,56).C. neoformans KN99a shows homogeneous filipin distribution on the cell membrane in the absence of BRI (Fig. 1g).By contrast, increased BRI concentrations induce the formation of filipin-stained patches on the cell membrane (Fig. 1g), suggesting BRI affects the ergosterol distribution and organization on the cell membrane.
To determine whether BRI displayed any interaction with sphingolipid, we combined various concentrations of BRI and myriocin, an inhibitor of serine palmitoyltransferase (Fig. 1d), the first step in glycosphingosine biosynthesis (57).Checkerboard assays showed FICs of 0.62 and 0.99 for both wild-type and erg3Δ strains, respectively, indicating an additive effect between BRI and myriocin (Fig. 1h).
We also examined the expression of genes involved in ergosterol biosynthesis (Fig. 1c) when C. neoformans was grown for 4 h in the presence of BRI (25 µM) or BRI + CAS (25 µM + 8 µg/mL).Compared to growth with no drug or CAS alone, ERG3 and SRE1 expression was induced by BRI about 10-to 15-fold, respectively, while ERG1 and ERG11 expression did not change (Fig. 1c and 2a).However, these levels of exposure to BRI did not modify the ergosterol content of the H99 wild-type and erg3Δ strains (notice that erg3Δ has lower ergosterol than the wild type in the absence of BRI; Fig. 1c and 2c).
The YSP2 gene of C. neoformans encodes a retrograde sterol transporter (52).Cells lacking this gene (ysp2Δ) accumulate ergosterol at the plasma membrane, leading to dramatic deformations of the membrane and cell wall.These mutant cells are also more sensitive than wild type to the sterol-targeting antifungal compound AmB and other membrane stressors (52).To measure the response of this mutant to BRI and CAS, we performed MIC assays, using YNB medium because ysp2Δ cells grow extremely poorly in RPMI (52).We found that the MIC for each compound was higher for ysp2Δ than for the corresponding wild type, KN99a: 32 versus 8 µg/mL for CAS (Fig. 2d) and 2.5 versus 1.25 µM for BRI (Fig. 2e).We noted that the MIC for CAS and BRI in KN99a strain grown in RPMI was higher, >32 µg/mL and 2.5 µM, respectively, than in YNB (see Table 1).Next, we tested for potential interactions of these compounds by performing checkerboard assays, again using YNB medium.These experiments indicated an additive effect, with no synergy or antagonism between the two drugs for either KN99a or ysp2Δ cells (Fig. 2f and  g).
Taken together, these results suggest that the primary BRI MoA in C. neoformans is perturbation of the cell membrane that reduces its organization and integrity, leading to increased membrane permeability and cell death.Although our results suggest that BRI can bind to free ergosterol, reduced sphingolipid concentration in the cell membrane also potentiates the MoA of BRI against C. neoformans.

BRI affects cell membrane organization, the CWI pathway, and calcium metabolism in S. cerevisiae
As an additional tool to investigate BRI MoA, we performed several screens using haploid null (ScWG), temperature-sensitive (TS), overexpression (MoBY), and heterozy gous (haploinsufficiency profiling, HET) strain libraries of the model yeast S. cerevisiae (16,58).Pooled cultures of barcoded S. cerevisiae strains from these collections were grown in liquid YPGal medium for 19 to 28 h at 26 or 30°C (Fig. 3a).The relative abundance of each barcoded strain following BRI treatment was then determined by high-through put sequencing of PCR-amplified barcodes followed by BEAN-counter analysis (59) to measure enrichment or depletion of each strain in the presence of BRI relative to solvent control (Tables S1 to S4; Fig. S1 to S4; https://doi.org/10.6084/m9.figshare.25239550).For all these analyses, we considered enriched or depleted log2 values of ≥2 or ≤−2, respectively.
We also examined combinations of antifungal compounds.As the BRI cannot inhibit S. cerevisiae growth at the highest concentration tested, the FIC value was not calcula ted.On the other hand, we used the SynergyFinder2.0(https://synergyfinder.fimm.fi) to calculate the interaction score between BRI and the other antifungal compounds.The interaction between the drugs was classified based on the synergy score where values lower than −10 were considered antagonistic, values from −10 to 10 was considered additive and values higher than 10 were considered synergistic.BRI potentiated azoles with cidal effects against S. cerevisiae, with scores of 23.854 and 23.852 for fluconazole and voriconazole, respectively, and additive synergy scores of 6.972 and −1.855 with CAS and anidulafungin, respectively (Fig. 3c through f).
Taken together, these results suggest that the MoA of BRI in S. cerevisiae involves ergosterol and sphingolipids biosynthesis, the CWI pathway, and calcium metabolism.

BRI potentiates AmB against C. neoformans
We have previously demonstrated that BRI potentiates CAS activity against C. neofor mans (40).Checkerboard assays showed that BRI + CAS had an additive interaction against C. neoformans strains H99 and KN99a and C. gattii strain R265 (FICs of 0.56, 0.56, and 0.76, respectively; Fig. 4a through c).By contrast, we did not observe any interaction between BRI + micafungin and BRI + anidulafungin (Fig. 4d and e).There is an additive interaction between BRI + AmB, BRI + voriconazole, BRI + fluconazole, and BRI + 5 -FC (Fig. 4d through i) against C. neoformans.CAS affects β-1,3-glucan biosynthesis activating cell wall assembly and remodeling, preventing resistance against osmotic forces, which then leads to cell lysis.However, the addition of different concentrations of sorbitol cannot improve C. neoformans growth in the presence of BRI (data not shown; BRI and different concentrations of sorbitol show an FIC index >1, indifference).

Casein kinases are important for BRI and CAS activities
To further assess the BRI MoA, a collection of 58 protein kinase inhibitors (PKI, at a concentration of 20 µM; Table S5; https://doi.org/10.6084/m9.figshare.25239550)was screened for effects on C. neoformans H99 growth and corresponding metabolic activity alone or together with BRI (1.25 µM) (Fig. 5a; Table S5  (SDS) treatment, osmotic and oxidative stresses, AmB, fluconazole, Congo red, calcofluor white, and regulates the phosphorylation of both Mpk1 and Hog1 mitogen-activated protein kinases during these stresses (61,62).Two independent cck1 (here called yck3∆) mutants in the C. neoformans H99 background were more sensitive than wild type to CAS and BRI + CAS but surprisingly not to BRI alone (Fig. 5d).The differences between the casein kinase PKIs potentiating BRI against C. neoformans and the lack of increased susceptibility of the yck3∆ mutants to BRI could be explained if (i) the three PKIs do not inhibit C. neoformans Yck3 or (ii) Cck2p is more important than Yck3 for the BRI mechanism of action.To address the first hypothesis, we incubated wild-type and yck3∆ mutant strains with the PKIs and assessed their viability (Fig. 5e).The yck3∆ mutants were more sensitive to the PKIs than the corresponding wild-type strain (Fig. 5e), emphasizing that C. neoformans casein kinases are targets for these compounds.Checkerboard assays confirmed an additive interaction between one of these PKIs (UNC-ALM-87) and BRI (Fig. 5f).We next examined the expression of genes involved in ergosterol synthesis in C. neoformans yck3Δ grown for 4 h in the presence of BRI and/or CAS.BRI (25 µM) and BRI + CAS (25 µM + 8 µg/mL) for 4 h induce about sevenfold more ERG1 than the wild-type strain in the control and in the presence of CAS with threefold reduction, but still higher levels than the wild type, in the presence of BRI (Fig. 2b).ERG3 and ERG11 showed no induction in the presence of the drugs but in the control, both gene levels are about 50% higher than in the wild-type strain (Fig. 2a and b).SRE1 expression is about 50% higher in the control, BRI and BRI + CAS than the wild-type strain, and additionally it was induced in the presence of CAS alone, which was not observed in the wild-type strain (Fig. 2a and  b).
Taken together, these results strongly suggest that casein kinases are involved in the BRI MoA and are important for the modulation of the expression of genes involved in ergosterol biosynthesis.

BRI potentiates CAS against C. neoformans through multiple mechanisms
To explore how BRI impacts the effect of CAS on C. neoformans, we examined the β-1,3-glucan synthase (Fks1) that is targeted by CAS.We observed without any further quantification that cell surface levels of Fks1:GFP were apparently lower in the pres ence of CAS-F (a functional fluorescently labeled probe (63); and BRI + CAS-F than in the control (Fig. 6a).We hypothesized that this reflected different levels of endocytic removal of the Fks1:GFP.When C. neoformans is treated either with CAS-F or BRI + CAS-F, both Fk1:GFP and CAS-F are internalized into small vesicles and structures that resemble vacuoles (Fig. 6a).The vacuoles were confirmed by co-staining with CellTracker Blue CMAC Dye (7-amino-4-chloromethylcoumarin), a hydrophobic molecule that enters the fungal cytoplasm and is modified through binding to glutathione by glutathione S-transferase (64).In fungi, CMAC accumulates in vacuoles, most likely through the transport of glutathione transporters present on the vacuolar membrane (64).Unfortu nately, in spite of several attempts, the resolution of the microscopy did not allow us to perform a differential analysis of the number of vesicles/vacuoles populated by CAS-F upon BRI treatment.However, our results suggest a possible internalization of Fks1:GFP and CAS-Fks1:GFP complexes by the C. neoformans endocytic machinery, followed by trafficking to the vacuoles.
To test this hypothesis, we selected a group of mutants lacking genes involved in the endocytic machinery (see Table 1 and references 65-67; for reviews, see references 68-70) and measured their MICs in the presence of BRI or CAS (Table 1).For these studies, we chose (i) two members of the CORVET membrane tethering complex (two independent null mutants of VPS3, encoding a cytoplasmic protein required for the sorting and processing of soluble vacuolar proteins, vps3-c7Δ and vps3-b8Δ, and two independent null mutants of VPS8, encoding a membrane-binding component of the CORVET complex, vps8-2Δ and vps8-38Δ), (ii) one member of the ESCRT-I complex (two independent null mutants of VPS23, involved in ubiquitin-dependent sorting of proteins into the endosome, vps23-9Δ and vps23-16Δ), (iii) one member of the ESCRT-III complex (SNF7, involved in the sorting of transmembrane proteins into the multivesicular body (MVB) pathway), (iv) two members of the HOPS endocytic tethering complex (two independent null mutants of VAM6, encoding a guanine nucleotide exchange factor for the GTPase Gtr1p, vam6-5Δ and vam6-89Δ) and VPS41 (encoding a vacuole membrane protein that functions as a Rab GTPase effector), (v) CHC1 (encoding a clathrin heavy chain, chc1-1Δ), and (vi) LAS17 (encoding an actin assembly factor, las17-4Δ).Apart from vps23Δ and snf7Δ, all mutants showed reduced MICs for BRI and CAS (Table 1).Check erboard assays showed that BRI and CAS have synergism and an additive interaction against C. neoformans vps3-8Δ and las17-4Δ mutant strains with FICs of 0.25 and 0.75, respectively (Fig. 6b and c).PI permeability into the cytoplasm is increased in the vps3-8Δ mutant when compared to the wild-type strains: BRI 1.25 and 2.5 µM, 10% versus 2 and 10%; CAS 8 µg/mL, 0% versus 2%; BRI + CAS (1.25 µM + 8 µg/mL), 25% versus 16%; and BRI + CAS (2.5 µM + 8 µg/mL), 65% versus 17% (Fig. 6d).Recently, C. neoformans CDC50, encoding a lipid flippase responsible for the translocation of phospholipids from the exocytoplasmic to the cytoplasmic leaflet, was shown to be important for CAS resistance.This loss leads to abnormal phospholipid distribution and impaired intracellular vesicular trafficking (36,37).We observed that cdc50Δ is not only more sensitive than wild type to CAS but also to BRI (Table 1).Checkerboard experiments with cdc50Δ showed synergism between CAS and BRI (FIC of 0.25; Fig. 6e).
Previously, we have shown that BRI acts in A. fumigatus in part by affecting the CWI pathway (40).We, therefore, tested the BRI and CAS susceptibility of a group of C. neoformans null mutants involved in the CWI pathway: (i) CNA1 (encoding a catalytic subunit of the phosphatase calcineurin), (ii) HOG1 and MPK1 (encoding mitogen-activa ted protein kinases from the high-osmolarity glycerol pathway and CWI, respectively), (iii) PKA1 (encoding the catalytic subunit of the protein kinase A), and (iv) RIM101 (encoding a transcription factor responsible for sensing extracellular pH signals) (for reviews, see references 71,72).All these mutants showed decreased CAS MICs, consistent with prior reports on cna1Δ and mpk1Δ (31,73).All except rim101Δ showed decreased MICs for BRI (Table 1).Checkerboard assays showed that BRI and CAS are synergistic against C. neoformans cna1Δ and mpk1Δ with FICs of 0.25 for both mutant strains (Fig. 6f and g).The cryptococcal cell wall is arranged in two layers that show different electron densities by transmission electron microscopy (TEM) (Fig. 7a).The inner layer is composed of β-glucans and chitin, mannoproteins and melanin, while the outer layer mainly contains α-and β-glucans.Treatment with CAS (8 µg/mL) led to increases in both the total cell wall and the inner layer, while BRI (25 µM) alone or BRI +CAS (25 µM + 8 µg/mL) yielded overall thickness similar to wild type but a marked reduction in the inner layer compared to both wild type and CAS treatment alone (Fig. 7a through c).To pursue these results, we examined the expression of genes involved in cell wall synthesis.We found that expression of FKS1 was induced about 10-, 4-, and 5-fold, respectively, when C. neoformans was grown in the presence of BRI, CAS, and BRI + CAS for 4 h (Fig. 7d).Expression of CHS3 (encoding a chitin synthase) was induced about 2.5-fold when C. neoformans was exposed to either BRI or BRI + CAS while expression of CHS4 and CHS6 (encoding chitin synthases) rose 100-, 2-, and 40-fold 2.5-, 7-, and 2.5-fold, respectively, in the presence of BRI, CAS, or CAS + BRI (Fig. 7d).Similar analysis of C. neoformans yck3Δ showed no induction of FKS1 with BRI, CAS, or BRI + CAS for 4 h although FKS1 expression was about 10-fold higher in the control than in the wild type (Fig. 7e); CHS3 showed about threefold induction in the presence of CAS but not BRI and BRI + CAS (Fig. 7e).CHS4 was induced in all treatments but about sevenfold more in the control than in the wild-type control, while CHS6 had about fourfold induction in the control than in the wild-type control, but induction only in the presence of CAS (Fig. 7e).
Next, we evaluated whether BRI or BRI + CAS could impact chitin distribution, organization, and exposure in the cell wall by staining it with the fluorescent dye Calcofluor White (CFW) (Fig. 7f).Exposure of C. neoformans wild type or yck3Δ cells to CAS for 8 h increased chitin staining on the cell surface, but this effect can be decreased by BRI and BRI + CAS (Fig. 7f).Interestingly, although chitin synthase levels are higher in the yck3Δ mutant than in the wild type, the chitin levels are lower in the mutant than in the wild type in all the treatments, except in the control, where the chitin levels are higher than in wild type (Fig. 7d through f).
Taken together, these results suggest that the endocytic machinery plays a role in the MoA of BRI + CAS combinations, affecting cell permeability.Moreover, the CWI pathway is also important for the BRI + CAS MoA, affecting mainly chitin accumulation in the cell wall.Finally, the casein kinase Yck3 is also important for the modulation of the expression of genes involved in cell wall construction.

BRI and BRI + CAS affect C. neoformans virulence
Next, we investigated whether BRI + CAS affected C. neoformans virulence and its virulence determinants, such as capsule formation and melanization.It has been speculated that the inefficacy of CAS in C. neoformans is because the cell wall or polysaccharide capsule prevents the accessibility of CAS to β-1,3-glucan synthase (31).We measure whether BRI, CAS, and BRI + CAS could inhibit both capsule size and formation (Fig. 8a through c).We found that CAS and BRI + CAS can equally inhibit the capsule diameter size and the number of capsular cells than the wild-type strain while there are no differences between BRI and the control (Fig. 8a through c).The C. neoformans acapsular mutants cap59Δ and cap67Δ were similarly BRI susceptible but more CAS-susceptible than the wild-type strain (Table 1).Checkerboard assays showed that BRI and CAS have additive effects against C. neoformans cap59Δ with an FIC of 0.75 (Fig. 8d).These results suggest that although BRI can potentiate more CAS activity against C. neoformans cap59Δ than the wild type, CAS affected capsule formation more than BRI.Also, we did not observe changes in C. neoformans melanin accumulation when the cells were exposed to BRI, CAS, and BRI + CAS either at 30 or at 37°C (Fig. 8e).
C. neoformans may be engulfed by host macrophages, potentially remaining latent inside of them or taking advantage of them for dissemination within the host (43).We found that BRI is not toxic to bone marrow-derived macrophages (BMDMs; Fig. 8f) and that BRI or BRI + CAS decreases C. neoformans survival in BMDMs about 80% to 90% (Fig. 8g).Considering the possibility that BRI changes the C. neoformans macrophage recognition due to possible changes in the capsule and cell wall previously reported, we also quantified kinetics of C. neoformans phagocytosis in the presence and absence of BRI 10 µM (Fig. 8h).In the absence of BRI, we observed phagocytic indexes of 24.6%, 33.3%, 7.2%, while in the presence of BRI 10 µM, phagocytic indexes of 17.8%, 15.4%, and 6%, in 1-, 2-, and 4-h phagocytosis, respectively (Fig. 8h).The phagocytic index in the presence of BRI in 2 h is significantly reduced 54% from the corresponding phagocytic time in the absence of BRI (Fig. 8h).These results suggest that BRI can affect both the initial steps of C. neoformans phagocytosis and initial and later steps of viability in the presence of BMDMs.
We also examined the host response to cryptococcal cell exposure in the presence of these compounds.CAS, BRI, and BRI + CAS are affecting the BMDMs tumor necrosis factor-alpha (TNF-α) production but did not change the levels of the anti-inflammatory cytokine IL-10 during C. neoformans BMDM infection (Fig. 8i and j).Importantly, BRI treatment BRI (5 mg/Kg, administered once daily by intraperitoneal injection for 13 days) can significantly decrease (about 34.5%) C. neoformans lung infection in an immunocom petent murine model of invasive pulmonary cryptococcosis (Fig. 8k).
Taken together, our results suggest that BRI has the potential to affect C. neoformans virulence and pathogenicity.

DISCUSSION
C. neoformans is an important fungal pathogen that affects a large global population of people with immunosuppressive conditions, including HIV patients.There are very few options to treat cryptococcal infections since Cryptococcus spp.are intrinsically resistant to echinocandins, and azoles are not very efficient because there are several mechanisms to develop azole resistance (16,19,74,75).The primary treatment for initial therapy in disseminated or central nervous system (CNS) cryptococcosis is AmB, which may be used alone or in combination with 5-FC.AmB has a rapid onset of action and often leads to clinical improvement more rapidly than either intravenous or oral fluconazole; after that, patients usually need to take fluconazole for an extended time to clear the infection.However, AmB causes substantial toxicity and its application is limited by economic issues (16).Recently, other drugs, such as fosmanogepix (which inhibits the GPI-anchor biosynthetic enzyme Gwt1), sertraline (a repurposed antidepressant that inhibits serotonin reuptake), tamoxifen (a repurposed breast cancer therapeutic that selectively modulates the estrogen receptor), and VT-1598 (an inhibitor of the ergosterol biosynthetic enzyme Erg11 that disrupts membrane integrity) are at different stages of clinical trial testing and are promising novel antifungal agents to treat cryptococcosis (for a review, see reference 16).Here, we demonstrate that the host defense peptide mimetic BRI is a novel compound with toxicity for not only reference strains and clinical isolates of C. neoformans but also C. gattii.BRI is efficient by itself but also can potentiate CAS and AmB against both species.BRI may thus offer a therapeutic alternative for cryptococcal infection.
Antimicrobial peptides directly or indirectly target microbial plasma membranes, disrupting their membrane potential (76).BRI has been shown to act by a similar mechanism in various bacterial species (54,77,78).We have previously shown that BRI can potentiate CAS and azoles against A. fumigatus, Candida albicans, C. auris, C. neoformans, and several species of mucorales (40).Here, we investigated the MoA of BRI and how BRI can potentiate CAS against C. neoformans.Using specific mutants and chemical inhibition, we demonstrate that BRI affects the organization of the C. neoformans cell membrane, influencing its permeability.Ergosterol is the major sterol in fungal cell membranes and is critical for the establishment of membrane fluidity and the regulation of cellular processes.Glycosphingolipids (GSLs) are key components of the plasma membrane and are involved in cellular processes crucial for fungi, such as growth, differentiation, signal transduction, and pathogenesis (53).GSLs are clustered along with sterols in specialized membrane microdomains termed lipid rafts, which are essential for cell membrane organization and crucial for the establishment of cell polarity (53,79).The organized construction of the cell membrane, with the appropriate distribution of GSLs and ergosterol, is needed for the correct assembly of essential cell membrane proteins as well as the fusion and deposition of vesicles containing precursors required for cell wall growth that are transported through a network of microtubules and the actin cytoskeleton (53,80).Our results suggest that BRI most likely affects the C. neoformans cell membrane organization by disrupting membrane potential and perturbing the distribution of ergosterol and sphingolipids.This will affect the cell homeostasis, accelerating the process of cell death.BRI may also influence the anchorage of the cell wall to the plasma membrane, affecting either cell wall morphology or its adherence to the plasma membrane (for reviews, see references [81][82][83].In both cases, altered concentrations of long-chain ceramides and complex sphingolipids could affect the rigidity of the cell wall and/or cell membrane (see reference 84; and for reviews, see references [81][82][83]. Changes in cell membrane composition and organization also affect protein secretion, endocytosis, and the proper deposition of components of the cell wall and plasma membrane (for a review, see reference 85).Consistent with this, we found that several mutants involved in endocytic traffic were more sensitive to BRI than wild-type cells and displayed increased permeability to PI when exposed to BRI.Membrane disorganization can also influence the correct organization and positioning of mem brane proteins, and membrane receptors that are essential for regulation of signaling transduction pathways essential for cell survival.We used the power of S. cerevisiae molecular genetics to identify gene products important for the cellular response to BRI.We observed a very complex multitarget MoA for BRI against S. cerevisiae, revealing multiple mutants affected in ergosterol and sphingolipid biosynthesis to be highly susceptible to BRI; other mutants important for cytoskeleton and cell wall biosynthesis were depleted or enriched after drug treatment.More interestingly, mutants lacking protein kinase C, MAP kinases from the CWI and HOG pathways, and calmodulin have altered survival upon BRI treatment, strongly indicating that BRI by itself is primarily influencing these pathways.
BRI impact is particularly evident when C. neoformans is exposed to CAS and there is an increase in the exposure of chitin or changes in the thickness in the C. neoformans cell wall that are repressed either in the presence of BRI or BRI + CAS.These effects are also observed on transcriptional regulation of genes important for the biosynthesis of different components of the cell wall.Our results indicate that BRI is hierarchically able to affect the cell membrane and cell wall organization.The observed CAS synergism could be related to an increasing worsening phenotype of cell wall disconstruction through the β-1,3-glucan depletion which will promote accentuated permeability and leakage of the cytoplasmic content and increased lethality observed upon BRI + CAS.This is further supported by our identification of casein kinase inhibitors when we screened for PKIs that could potentiate BRI.The nonessential C. neoformans casein kinase 1 cck1/yk3 (61,62) has several deficient phenotypes related to cell membrane homeostasis and the cell wall integrity pathway and regulates the phosphorylation of both Mpk1 and Hog1 mitogen-activated protein kinases (MAPKs) during cell wall and cell membrane stresses (61,62).Comparatively, the Candida albicans casein kinase 1 family member yck2Δ/yck2Δ mutant was hypersusceptible to cell wall damaging agents, had increased chitin content in the cell wall, and increased mRNA accumulation of the chitin synthase genes, CHS2, CHS3, and CHS8 (86).A screening of protein kinase inhibitors to reverse C. albicans CAS resistance identified a compound able to restore CAS sensitivity whose target is Yck2 (60).Recently, a screening for C. albicans kinome to identify genes for which loss-of-func tion confers hypersensitivity to echinocandins and azoles revealed the casein kinase 1 (CK1) homolog Hrr25 as a regulator of tolerance to both antifungals and especially important as a target-mediated echinocandin resistance (87).BRI potentiates the action of CAS against C. albicans (40), but it remains to be determined if C. albicans casein kinase mutants have increased BRI susceptibility.C. neoformans Cck1/Ypk3 modulates the expression at the transcriptional level of genes encoding ergosterol biosynthesis, β-1,3-glucan synthase, and chitin synthase.Other C. neoformans protein kinases that interact with casein kinases, such as calcineurin and MAP kinases, also have decreased BRI susceptibility.
When we investigated the influence of BRI on several key C. neoformans virulence determinants, we found that BRI + CAS decreases capsule formation but not melanin production.BRI also significantly reduces C. neoformans phagocytosis and survival inside macrophages, despite being nontoxic to BMDMs.It partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis.Interestingly, CAS and BRI can increase the TNF-α but not the interleukin-10 (IL-10) cytokines production.TNF-α plays a critical role in the control of C. neoformans and its lack stimulates its persistence (88) while IL-10 signaling has been shown as important for persistent cryptococcal lung infection (89).These results indicate that BRI can immuno modulate cytokine production and decrease the C. neoformans infection.
We have demonstrated that BRI is potentially an important antifungal agent against cryptococcosis, particularly important for synergizing with CAS, which is otherwise ineffective against this pathogen.Future work will determine how BRI interacts physically with components of the cell membrane, and how this affects CWI.

Strains, media, and cultivation methods
All the Cryptococcus strains are shown in Table 1.For all studies, C. neoformans and C. gattii strains were inoculated from single colonies into YPD media (2% [wt/vol] dextrose, 2% [wt/vol] Bacto peptone, and 1% [wt/vol] yeast extract in distilled water [dH 2 O]) and grown overnight at 30°C with shaking at 160 rpm before further handling as detailed below.Agar was added to a final concentration of 1.5% when solid medium was used.RPMI-1640 medium (Gibco) supplemented with 9.6 mM HEPES (Sigma-Aldrich) was used for metabolic activity assays and MIC assays.

Minimal inhibitory concentration
The BRI drug used for MIC assays was kindly supplied by Innovation Pharmaceuticals Inc. and solubilized in DMSO.The MIC for Cryptococcus strains was determined based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (90), using the E246-7 method.In brief, the MIC assay was performed in 96-well flat-bottom polystyrene microplates where 100 µL of a 10 5 cells/mL stock prepared in liquid RPMI-1640 media was dispensed in each well and supplemented with increasing concentration of drugs.Plates were incubated at 30°C for 48 h and the inhibition of growth was evaluated.The MIC was defined as the lowest drug concentration that yielded 100% fungal growth inhibition, assessed visually, compared with control wells containing only RPMI-1640 medium and DMSO.

Culture conditions and ergosterol extraction
The extraction and quantification of ergosterol were carried out as described previously with some modifications (91).Cryptococcus strains (KN99a and erg3Δ) were grown in liquid YPD medium at 30°C with shaking for 16 h.Next, cells were collected by centrifuge (3,000 rpm for 10 min) and washed with sterile distilled water.Cells were counted by hematocytometer and the inoculum was adjusted to 10 7 to 10 8 cells into liquid RPMI-1640 medium with (2.5 µM or 25 µM) or without brilicidin.The cells were incubated at 30°C for 4 h and then collected and washed as above.Three milliliters of 25% ethanolic of KOH solution was added to each pellet and vortexed for 1 min.The cell suspension was transferred to borosilicate glass screwcap tubes to incubate at 85°C for 1 h in a water bath.Sterols were extracted by adding a mixture of 1 mL of sterile distilled water and 3 mL of petroleum ether.The petroleum ether layer was transferred to a clean borosilicate glass and sterols were detected by reading absorbance at 260 nm and 280 nm.

Checkerboard assays
For measuring the effect of the combination between BRI and antifungal drugs against Cryptococcus spp., metabolic activity by XTT assay was used (92).A concentration of 10 5 cells/mL was inoculated in liquid RPMI-1640 supplemented with increased concen trations of BRI (x-axis) and increased concentrations of CAS, 5-fluorocytosine (5-FC), FLC, latrunculin-B, or AmB (y-axis) in 96-well flat-bottom polystyrene microplate.The plates were incubated at 30°C for 48 h.The viability of cells was revealed using XTT assay as described (92).Before the XTT assay, 3 µL from each well was collected and plated on solid YPD for cidality evaluation.The plates were incubated at 30°C for 24 h and photographed.To determine synergy, additive, indifference, or antagonism, we used the FIC index method (93), where an FIC index of <0.5 indicates synergism, 0.5-4 indicates additive or indifference, and >4 is considered to be antagonism (94).
For S. cerevisiae, the interaction between BRI (0-80 µM) and fluconazole (0-16 µg/mL), voriconazole (0-4 µg/mL), CAS (0-1 µg/mL), or anidulafungin (0-2 µg/mL) was assessed through a checkerboard assay using liquid YPGal medium.Briefly, single colonies of cells were inoculated from solid YPD in liquid YPD and incubated for 16 h at 30°C.Cells were washed twice in liquid YPGal and resuspended to a concentration of 2.5 × 10 3 cells/mL in the same medium supplemented with increasing concentrations of BRI (x-axis) and increasing concentrations of fluconazole, voriconazole, CAS, and anidulafungin in 96-well flat-bottom polystyrene microplate.The plates were incubated at 30°C for 48 h and the metabolic activity was measured using XTT assay as described (92).Before the XTT assay, the cidality was evaluated by plating 3 µL from each well on solid YPD.The plates were incubated at 30°C for 24 h and photographed.We used SynergyFinder2.0(https://synergyfinder.fimm.fi) to calculate the interaction score between BRI and the other antifungal compounds.The interaction between the drugs was classified based on the synergy score where values lower than −10 were considered antagonistic, values from −10 to 10 were considered additive and values higher than 10 were considered synergistic.
For evaluation of membrane integrity, the H99 and KN99a strains and the mutants vps3∆ and erg3∆ were incubated in liquid RPMI-1640 media under control conditions or treatment with BRI (1.25, 2.5, 12.5, and 25 µM), CAS (8 µg/mL), or the combination of BRI + CAS and incubated at 30°C for 1 h, 2 h, or 4 h.Then, the cells were washed two times with 1× PBS and staining in 1× PBS with propidium iodide (10 µg/mL 15 min, RT) or Filipin (10 µg/mL 20 min, RT).The cells were collected and washed three times with 1× PBS.
Slides were visualized on an Observer Z1 fluorescence microscope using a 100× objective oil immersion lens for Filipin, filter set 38-high efficiency (HE), excitation wavelength of 450-490 nm, and emission wavelength of 500-550 nm; and for PI, the wavelength excitation was 572/25 nm and emission wavelength was 629/62 nm, filter set 63 HE.Ddifferential interference contrast (DIC) images and fluorescent images were captured with an AxioCam camera (Carl Zeiss) and processed using AxioVision software (version 4.8).In each experiment, at least 50 cells were observed and the experiment was repeated at least two times.

Transmission electron microscopy
C. neoformans strain KN99a was grown overnight in YPD as above, washed in distilled water, resuspended at 10 8 /mL in RPMI-1640 medium (Gibco), and dispensed (1 mL aliquots) into 24-well plates.Caspofungin diacetate and brilacidin tetrahydrochloride were added to achieve the indicated final concentrations and the plates were incubated (8 h, 30°C) before cell fixation with 2% paraformaldehyde/2.5% glutaraldehyde (Ted Pella Inc., Redding, CA) in 0.1 M sodium cacodylate buffer (2 h at room temperature and then overnight at 4°C).Samples were next washed in sodium cacodylate buffer and post-fixed in 1% osmium tetroxide (Ted Pella Inc.) for 1 h at room temperature.After three washes in distilled water, samples were en bloc stained in 1% aqueous uranyl acetate (Electron Microscopy Sciences, Hatfield, PA) for 1 h, rinsed in distilled water, dehydrated in a graded series of ethanol, and embedded in Eponate 12 resin (Ted Pella Inc).Ultrathin sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) equipped with an AMT 8megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques, Woburn, MA).

Staining for cell surface components
Cell wall surface polysaccharide staining was performed using at least six repetitions.Briefly, the cells were grown overnight in YPD.Then, washed with PBS and 100 µL of 10 6 cells/mL suspension was inoculated in RPMI-1640 medium with BRI (25 µM) and/or CAS (8 µg/mL), in a 96-well microplate.The plate was incubated for 8 h at 30°C.The culture medium was removed and the cells were fixation with PBS 3.7% formaldehyde/2.5% glutaraldehyde for 5 min at RT.For chitin staining, 100 µL of PBS with 25 µg/mL of calcofluor white (CFW) was added to the wells, which were incubated for 10 min at RT and washed twice with PBS before fluorescence was read at 380 nm excitation and 450 nm emission.The fluorescence was read in a microtiter plate reader (SynergyHTX Multimode Reader; Agilent Biotek or EnSpire Multimode Plate Reader; Perkin Elmer).

RNA isolation, cDNA synthesis, and RT-qPCR analysis
All experiments were carried out in biological triplicates, C. neoformans H99 and yck3∆ strains were grown overnight in YPD as above.Cells were washed in PBS and resuspen ded at 10 6 cells/mL in RPMI-1640 medium.BRI (25 µM) and CAS (8 µg/mL) were added and the inoculum was incubated for 4 h at 30°C, 180 RPM.The cells were washed in PBS and the samples were lyophilized.A total RNA was isolated by TRIzol (Invitrogen) after cellular lysis by glass beads and treated with RQ1 RNase-free DNase I (Promega).RNA integrity and concentration were assessed using a NanoDrop Lite Spectrophotom eter (Thermo Scientific).For RT-qPCR, the RNA was reverse transcribed to cDNA using the ImProm-II reverse transcription system (Promega) according to the manufacturer's instructions, and the synthesized cDNA was used for real-time analysis using the SYBR green PCR master mix kit (Applied Biosystems) in the ABI 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA).Sybr Primer sequences are listed in Table S6 (https://doi.org/10.6084/m9.figshare.25239550).ACT1 (actin-1) gene was used as normalizer.

Transcription factor and protein kinase inhibitor screening
To identify possible genes involved in the mechanism of BRI action against Cryptococcus spp., we screened a library of C. neoformans H99 mutants for transcription factors.For screening the library, the frozen cells were inoculated in 600 µL liquid YPD in a deep-well plate for 48 h at 30°C, 180 rpm.After incubation, 100 µL of each well was transferred to a clear 96-flat bottom-plate and optical densities measured at 600 nm; cells were normalized to an OD 600 of 1 in a final volume of 100 µL YPD.Then, cells were diluted 1:20 in 200 µL liquid RPMI-1640 and adjusted to a final concentration of 10 5 cells/mL in liquid RPMI-1640 media supplemented with BRI (1.25 µM) or CAS (8 µg/mL), in two replicate plates.Fresh liquid RPMI-1640 with DMSO was used as a control.The plates were incubated at 30°C for 48 h.The absorbance was read at 600 nm to determine the optical density and 3 µL from each well was plated on solid YPD to evaluate cell viability.
The candidates initially selected as sensitive to BRI or CAS were grown overnight in liquid YPD at 30°C, 180 rpm.Then, cells were washed twice with 1× PBS and cellular density was adjusted to 10 6 cells/mL.A microdilution was performed and the cells were grown in a 96-flat bottom-plate on liquid RPMI-1640 media in the presence (or not) of BRI (1.25 µM) or CAS (8 µg/mL) for 48 h at 30°C.After that, 3 µL from each well was plated on solid YPD and incubated at 30°C for 24 h.Cell viability was assessed visually and photographed.
A protein kinase inhibitor (PKI) library was also screened in combination with BRI.In total, 58 PKI were analyzed (Table S5; https://doi.org/10.6084/m9.figshare.25239550).Briefly, 100 µL of liquid RPMI-1640 containing 10 5 cells/mL of the C. neoformans H99 strain was inoculated with PKI (20, 40, and 60 µM) in the presence (or not) of BRI (1.25 µM) and incubated 48 h at 30°C.The cells were then collected and plated on solid YPD for CFU counting.Experiments were repeated at least two times.

Luria-Delbruck fluctuation assay
Fluctuation assays were conducted as previously described (96).Briefly, 10 independent H99 colonies were selected from a YPD agar stock and cultured overnight in 5 mL liquid YPD at 30°C.The cultures were washed 2× with dH 2 O, resuspended in 5 mL dH 2 O, and plated onto the appropriated solid RPMI medium (100 µL of cells at a 10 −5 dilution were plated onto RPMI, and 100 µL undiluted cells onto RPMI + 5 FC [100 µg/mL] and RPMI-1640 + BRI [100 µM]; no repeated measurements).The plates were incubated at 37°C; colonies were counted following incubation for 4 (control RPMI-1640 medium) or 14 days (drug media).Data from the fluctuation assay were analyzed using the R package flan v0.9 (97).The analysis was conducted with default parameters, employing a 95% confidence interval for the mutation probability and the maximum likelihood method.

Capsule formation analysis
To qualitatively assess capsule thickness, H99 strains were grown on YPD medium for 16 h and washed with PBS, and 10 5 cells were incubated in Dulbecco's MEM (Gibco) with 10% FBS, in a control condition and the presence of BRI (1.25 µM) or CAS (8 µg/mL), and the combination of BRI + CAS (1.25 µM and 8 µg/mL, respectively) for 72 h at 37°C and 5% CO 2 .Cells were collected and counter-stained with India ink (1:1) for microscopy analysis.Relative capsule sizes were defined as the ratio between the capsule thickness and cell diameter.ImageJ software was utilized to determine the capsule measurements of 50 cells of two technical replicates.

Macrophage culture
BALB/c BMDMs were obtained as previously described (99).Briefly, bone marrow cells were cultured for 7-9 days in Dulbecco's modified Eagle medium (DMEM) 20/30, which consists of DMEM (Gibco, Thermo Fisher Scientific Inc.) supplemented with 20% (vol/vol) FBS and 30% (vol/vol) L-cell conditioned media (LCCM) as a source of macrophage colony-stimulating factor (M-CSF) on non-treated petri dishes (Optilux-Costar, Corning Inc. Corning, NY).Twenty-four hours before experiments, BMDMs were detached using cold phosphate-buffered saline (PBS) (Hyclone, GE Healthcare Inc.South Logan, UT), the cell viability and concentration were adjusted using a hemocytometer as previously described (100) diluting 1 part of 0.4% trypan blue (Gibco) and one part cell suspension.Then, the unstained (viable) and stained (nonviable) cells were counted separately, and the percentage of viable cells was calculated as follows: viable cells (%) = total number of viable cells per mL/total number of cells per mL × 100.
For functional assays, 1 × 10 6 cells were added to 24-well tissue culture plates containing the differentiation Eagle medium (DMEM) 20/30 and allowed to grow adherently overnight at 37°C.Non-adherent cells were removed by gently washing three times with warm PBS.The viability of the BMDMs preparations was >99% as judged by trypan blue dye exclusion and the cells percentage is greater than 90% differentiated macrophages.

Macrophage internalization and killing assay
Bone marrow-derived macrophages were prepared as described above.Briefly, 10 6 cells/ well were seeded in a 24-well plate and incubated in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 for 24 h.C. neoformans cells were prepared for uptake experiments by inoculating an overnight culture in YPD and growing at 30°C, 180 rpm.To initiate the study, cryptococcal cells were washed with PBS and opsonized with anti-glucuronoxylomannan antibody 18B7 (1 µg/mL, Sigma) for 1 h at 37°C, while macrophages were activated with 1 µg/mL liposaccharide (LPS) for 30 min at 37°C and 5% CO 2 ; 10 7 cryptococcal cells were then incubated with the macrophages at 37°C and 5% CO 2 .After 24 h, the wells were washed three times with warm 1× PBS, and the macrophages were lysed with dH 2 O with 0.1% Triton X-100.This suspension was then diluted and 25 µL was plated on solid YPD and incubated at 30°C for 24 h for CFU counting.Also, the cell supernatant was collected and reserved for cytokine quantification assays.

Phagocytosis assay
Bone marrow-derived macrophages were prepared as described above.Briefly, 5 × 10 5 cells/well were seeded in a 24-well plate containing circular glass coverslips and incubated in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 for 24 h.C. neoformans cells were prepared for uptake experiments by inoculating an overnight culture in YPD and growing at 30°C, 180 rpm.To initiate the study, cryptococ cal cells were washed with PBS and opsonized with anti-glucuronoxylomannan antibody 18B7 (1 µg/mL, Sigma) for 1 h at 37°C.After the incubation, the cells were washed and incubated with 0.1 mg/mL FITC (Sigma) in 0.1 M Na 2 CO 3 at 37°C for 30 min.While macrophages were activated with 1 µg/mL liposaccharide (LPS) for 30 min at 37°C and 5% CO 2 ; labeled cells were washed three times with PBS, and 5 × 10 6 cryptococcal cells were then incubated with the macrophages at 37°C and 5% CO 2 .After 2 h, labeling of extracellular cryptococcal cells was performed by incubation with PBS, 0.25 mg/mL calcofluor white (Sigma) for 30 min at 4°C.The cells were washed twice with PBS and fixed with 3.7% (vol/vol) formaldehyde/PBS for 15 min followed by two washes with PBS.Microscopic photographs were taken on a Zeiss microscope.

Animal procedures
Inbred female mice (BALB/c strain; body weight, 20-22 g; obtained from the facility of the campus of the University of São Paulo, Ribeirão Preto, Brazil) were used in this study.Mice were housed in five per cage and had access to food and water ad libitum.Cages were well ventilated, softly lit, and subjected to 12:12 h light-dark cycle.The relative humidity was kept at 40%-60% and mouse room cages were kept at 22°C.The C. neoformans H99 strain was sub-cultured at 30°C for 48 h on a solid YPD medium.Prior to inoculation, colonies were taken from the subculture and inoculated into liquid YPD and grown overnight at 30°C, 180 rpm; cells were collected by centrifugation and washed three times in PBS.Mice (10 mice per group) were anesthetized by halothane inhalation and infected by intranasal instillation of 20 µL 10 6 cells of C. neoformans H99.The viability of the administered inoculum was determined by incubating a serial dilution of the cells in solid YPD, at 30°C.Treatment groups will consist of a vehicle (Cavitron W7 HP7 from Ashland, day 1-13) and BRI (10 mg/kg, day 1; 5 mg/kg, day 2-13).The drugs used in the mice treatment were prepared on day 1 and again on day 7, and stock refrigerated.
For vehicle solution preparation, 20% Cavitron W7 HP7 was diluted in sterile water for injection and filtered in 0.22 µm sterile filters.For brilacidin treatment, stock dosing solutions were prepared from the received brilacidin tetrahydrochloride dry powder (Innovation Pharmaceuticals), diluted in vehicle solution, and filtered in 0.22 µm sterile filters.The drugs were prepared to deliver their respective dose within a 0.1 mL volume.Animals (10 per group) were treated for 13 days (started on the day 1 post-infection) once daily by intraperitoneal (IP) injection.Mice were sacrificed 14 days post-infection.Animals were clinically monitored at least twice daily.Any animal that appears moribund prior to the scheduled endpoint was humanely euthanized.As a negative control, a group of 10 mice received vehicle only.To investigate fungal burden, the lungs were harvested and stored in ice.Samples were homogenized with sterile PBS with protease inhibitor cocktail tablets complete tablets, mini EDTA-free (Roche) and homogenates adequately diluted for fungal burden evaluation by colony forming units.

Cytokine quantification
ELISA was used to quantify the cytokine levels in cells supernatant, after 24 h of exposure to C. neoformans H99.The quantification of cytokines tumor necrosis factor-alpha and IL-10 was performed according to the manufacturer's instructions, using ELISA-assay kits (R&D Systems).The plate's final absorbance was read at 450 nm and the cyto kine concentration analysis (pg/mL) was performed according to the manufacturer's instructions, considering the values obtained in the standard curve of each evaluated cytokine.

Statistical analysis
Grouped column plots with standard deviation error bars were used for representations of data.For comparisons with data from wild-type or control conditions, we performed one-tailed, paired t-tests or one-way analysis of variance (ANOVA).All statistical analyses and graphics building were performed using GraphPad Prism 8 (GraphPad Prism Software).advice and materials for the work.G.H.G. and C.D. analyzed the data and wrote the manuscript, and G.H.G. coordinated all the work.All the authors read and edited the manuscript.

FIG 1
FIG 1 Ergosterol and glycosphingolipids are important for the BRI MoA against C. neoformans.(a) Fluctuation assays for C. neoformans grown on solid RPMI medium containing 100 µg/mL 5-FC or 100 µM BRI.(b) C. neoformans wild-type and null mutant strains were grown at 48 h in RPMI ± BRI (1.25 µM) and plated on solid YPD.(c and d) Diagrams showing the ergosterol and glycosphingolipid biosynthesis pathways in fungi.(e) Percentage of cells with propidium iodide (PI) accumulation in the C. neoformans KN99a strain.Notice the % on the top of the bars represent the cell viability upon exposure to different BRI concentrations.The results shown are the average of two repetitions with 50 cells each ± standard deviation.Statistical analysis: ordinary one-way ANOVA **P value < 0.005; ****P value < 0.0001.(f ) XTT assays for C. neoformans strains grown for 48 h at 30°C with the indicated concentrations of ergosterol and BRI.(g) C. neoformans KN99a strain was exposed to the indicated concentrations of BRI for 4 h and stained with filipin.(h) Checkerboard and cidality assays for C. neoformans KN99a and erg3Δ strains grown in RPMI with the indicated concentrations of myriocin and BRI.

FIG 2 C
FIG 2 C. neoformans retrograde sterol transporter ysp2Δ leads to abnormal accumulation of ergosterol at the plasma membrane and it is less susceptible to CAS and BRI.(a) RTqPCR for ERG1, ERG3, ERG11, and SRE1 mRNA accumulation when C. neoformans H99a wild type was grown for 16 h and transferred or not to either BRI (25 µM), CAS (8 µg/mL), or BRI + CAS (25 µM + 8 µg/mL).(b) RTqPCR for ERG1, ERG3, ERG11, and SRE1 mRNA accumulation when C. neoformans yck3Δ was grown for 16 h and transferred or not to either BRI (25 µM), CAS (8 µg/mL), or BRI + CAS (25 µM + 8 µg/mL).The results are the average of three repetitions ± standard deviation.Statistical analysis: two-way ANOVA ****P value < 0.0001.(c) Ergosterol concentration in the C. neoformans H99a wild type and erg3Δ mutant.(d and e) Wild-type strain (KN99a) or ysp2Δ cells were grown in YNB (pH 7.4) for 72 h at 37°C with CAS (c) or BR.(d) and plated for viability.Mean ± SE values of colony forming units (CFUs) per well are plotted; shown is one representative study of three independent experiments performed in technical triplicate.Statistical analysis: unpaired Student's t-test *P < 0.05; **P < 0.005.(f and g) The same strains were grown in YNB (pH 7.4) for 48 h at 30°C in the presence of the indicated concentrations of CAS and BRI.Metabolic activity measured by XTT assay is shown.ΣFIC, combined fractional inhibitory concentration; black boxes, Abs 492 > 1.0.

FIG 3
FIG 3 In S. cerevisiae, BRI affects cell membrane organization, cell wall integrity pathway and calcium metabolism.(a) S. cerevisiae pooled haploid null (ScWG), heterozygous (HET), temperature sensitive (TS), and overexpression (MoBY) libraries were grown in liquid YPGal medium in the absence or presence of BRI for 19 h at 30 o C (ScWG and HET, 6.25 to 100 µM BRI), 28 h at 26 o C (TS, 1.5625 to 25 µM BRI), or 24 h at 30 o C (MoBY, 20 to 100 µM BRI).(b) Categorization of the hits for each library according to Gene Ontology Term Finder (https://www.yeastgenome.org/goTermFinder)using a P-corrected value < 0.001.The number of engineered genes is indicated for each library as red for depleted or green for enriched.(c) Heat map of the genes that correspond to the mutants depleted or enriched in the presence of BRI and visually inspected as more modulated.(d-g) Checkerboard and cidality assays for S. cerevisiae grown in YPGal with the indicated concentrations of BRI + fluconazole, BRI + voriconazole, BRI + CAS, and BRI + anidulafungin.

FIG 5
FIG 5 Casein kinases are involved in BRI MoA.(a) C. neoformans growth screening of 58 PKIs (20 µM) in the absence or presence of BRI (1.25 µM).Cells were grown for 48 h at 30°C in liquid RPMI medium and plated on solid YPD medium.(b) Reduction of metabolic activity by PKIs in the absence or presence of BRI.The cells were grown for 48 h at 30°C in RPMI medium and metabolic activity was measured using XTT.(c) Viability of C. neoformans growth with or without PKIs for 48 h at 30°C.(d) Viability of C. neoformans wild type and yck3Δ mutants after incubation for 48 h at 30°C in RPMI medium in the absence or presence of various concentrations of BRI and/or CAS.(e) Viability of C. neoformans wild type and yck3Δ mutants after incubation for 48 h at 30°C in RPMI medium in the absence or presence of PKIs.Statistical analysis: two-way ANOVA *P value < 0.05; **P value < 0.01; ***P value < 0.001; ****P value < 0.0001 (f ) Checkerboard assay using XTT.C. neoformans was grown for 48 h in RPMI medium with various combinations of UNC-ALM-87 and BRI.

FIG 6
FIG 6 Potentiation of CAS by BRI against C. neoformans depends on the endocytic pathway.(a) C. neoformans Fks1:GFP was grown for 16 h in YPD media at 30°C and incubated in RPMI medium with or without CAS-F (fluorescent caspofungin) or BRI + CAS-F and staining with CMAC, CellTracker Blue CMAC Dye (7-amino-4-chloromethylcoumarin).(b and c) Checkerboard and cidality assays using XTT.C. neoformans vps3-8Δ (b) and las17-4Δ (c) were grown for 48 h at 30°C with various concentrations of BRI and CAS.(d) Percentage of cells with PI accumulation in C. neoformans H99 and vps3-8Δ strains.The cells were exposed to different concentrations of BRI for 4 h and cells with PI accumulated in the cytoplasm were counted.The results are the average of two repetitions with 50 cells each ± standard deviation.(e-g) Checkerboard and cidality assays using XTT.C. neoformans cdc50Δ (e), cna1Δ (f ), and mpk1Δ (g) were grown for 48 h at 30°C with different concentrations of BRI and CAS.

TABLE 1 C
. neoformans and C. gattii MICs